Method of pre-sensitizing cancer prior to treatment with radiation and/or chemotherapy and a novel cytokine mixture

ABSTRACT

This invention relates to a breakthrough method for pre-sensitizing cancer prior to a therapeutic treatment such as chemotherapy, radiation therapy or immuno-therapy and a novel cytokine mixture used in the method thereof The cytokine mixture is a serum-free and mitogen-free mixture comprised of specific ratios of cytokines such as IL-1β, TNF-α, IFN-γ and GM-CSF to Interleukin 2 (IL-2), which is effective in inducing cancerous cells to enter a proliferative cell cycle phase thereby increasing their vulnerability to chemotherapy, radiation therapy and immuno-therapy. One such novel cytokine mixture is Multikine®, which can be used alone or in combination with other drugs for the treatment of cancer thereby increasing the success of cancer treatment and the disease free survival of cancer patients.

INTRODUCTION

This invention relates to a breakthrough method for pre-sensitizingcancer prior to a therapeutic treatment such as chemotherapy, radiationtherapy or immuno-therapy and a novel cytokine mixture used in themethod thereof. The cytokine mixture is a serum-free and mitogen-freemixture comprised of specific ratios of cytokines such as IL-1β, TNF-α,IFN-γ and GM-CSF to Interleukin 2 (IL-2), which is effective in inducingcancerous cells to enter a proliferative cell cycle phase therebyincreasing their vulnerability to chemotherapy, radiation therapy andimmuno-therapy. One such novel cytokine mixture is Multikine®, which canbe used alone or in combination with other drugs for the treatment ofcancer thereby increasing the success of cancer treatment and thedisease free survival of cancer patients.

BACKGROUND OF THE INVENTION

Current treatments of cancer and in particular solid tumors, comprisemainly of surgical intervention followed by radiation therapy and/orchemotherapy. Dunne-Daly CF, “Principles of radiotherapy andradiobiology”, Semin Oncol Nurs. November 1999; 15(4):250-9; Hensley M Let al., “American Society of Clinical Oncology clinical practiceguidelines for the use of chemotherapy and radiotherapy protectants.”, JClin. Oncol. October 1999; 17(10):3333-55. In conjunction with suchtherapies, toxic chemotherapeutic agents such as Gemcidabin,Vinblastine, Cisplatin, Fluorouracil, Gleevec, Methotrexate, which areunable to differentiate between normal and cancerous cells, are used.While effective, these and other toxic chemotherapeutic agents have donelittle to increase overall patient survival. Moreover, currenttreatments in general also failed to improve 5-year survival rate ofcancer patients despite synergistic combination of chemotherapies andradiotherapies. Even anti-epidermal growth factor receptor agents,anti-angiogenic drugs and immuno and immuno-adjuvant therapy using drugssuch as Rituximab, Erbitux and Herceptin have failed to significantlyincrease the 5-year survival rate of cancer patients. Furthermore,complete remission or disease free survival of cancer patientirrespective of cancer type have not been improved upon by any of theaforementioned therapies or synergistic combinations thereof.

One treatment modality investigated to improve disease-free survivalrates or lead to complete remission is manipulation of cell-divisioncycle of cancerous cells. In particular, cycling tumor cells aregenerally more vulnerable to radio- and chemotherapies than non-cyclingtumor cells because complex biochemical and biomolecular processes suchas enzyme-dependent DNA replication, enzyme-dependent phosphorylation,signal cascades, association and dissociation of transcriptionalactivating molecular complexes, and formation and dissociation ofmacromolecular assemblies of cytostructural elements are required duringcell cycling. By inducing tumor cells into a cell cycle phase,anti-metabolic agents that inhibit any of the complex biochemicalprocesses such as ribonucleotide reductase (RNR) inhibitors,dihydrofolate reductase inhibitors or DNA polymerase inhibitors can beused to stop cell cycling and thereby prevent tumor proliferation.

However, known methods taking advantage of cell cycling are limited tosynchronizing cell cycle arrest with sequential applications of achemotherapeutic agent. For example, one known method arrests malignantcells within a S phase of the cell cycle with pyrimidine analogsfollowed by exposure to high concentrations of anti-metabolites. B.Bhutan et al., Cancer Res. 33:888-894 (1973). Few or no cells in thepopulation can proceed beyond this point of detention after applicationof the anti-metabolite. W Vogel et al., Hum. Genet. 45:193-8 (1978).

Other efforts include methods of manipulating the cell-division cycle byaltering the cell cycle distribution within the cell population. Theseprotocols stimulate malignant cells from a dormant phase into a cellcycling phase thereby increasing their vulnerability to anti-metabolicdrugs acting during the vulnerable DNA replication phase. H H Euler etal., Ann. Med. Interne. (Paris) 145:296-302 (1994); B C Lampkin et al.,J. Clin. Invest. 50:2204-14 (1971); Alama et al., Anticancer Res.10:853-8 (1990). Conversely, other known methods prohibit normal cellsfrom entering S phase thereby protecting normal cells from chemotacticdrugs.

Still another known method of synchronizing cell cycle phase withchemotherapeutics is a so-called pulse dose chemotherapy described by RE Moran et al., Cancer Treat. Rep. 64:81-6 (1980). In this approach,leukemic tumor cells in mice were detained in a S phase of the cellcycle with an infusion of hydroxyurea. After the infusion, the cellswere “released” to continue cell cycling wherein a “pulse” of a secondagent (Ara-C) was given to the mice. The intent was to maximize impactof the second agent as the cycling cells were moving through thevulnerable cell cycle S-phase. However, the results indicated that whilemice treated with Ara-C just after the hydroxyurea infusion showedimproved survival, mice treated with Ara-C at later times after thehydroxyurea infusion did not show improved survival. Clearly, simplysynchronizing cell cycling with a second agent acting non-simultaneouslydid not improve the action of the two agents.

Nevertheless, known methods taking advantage of cell cycle continue toseek an optimal but passive synergy between dosage, pharmacokinetics,sequence and scheduling.

It might be expected that confining a cell population to a vulnerablecell cycle phase where cells are specifically vulnerable to damage mightshift the dynamics of cell killing toward greater efficiency with agreater reduction in side-effects by diminishing exposure to toxicdrugs. However, actual experiments taking advantage of cell cycle arrestor static synchronization have been disappointing because known methodsare unable to actually induce cells into a cell cycle. Rather, all theknown methods simply time the synergistic combination of cell cyclearrest or static synchronization with the target cell population.Moreover, agents such as pyrimidine and hydroxyurea used to effect thecell cycle can cause damage to normal cells.

Another approach would, of course, be to induce the cells to enter intoa cell cycle phase as opposed to arresting the cell cycle orsynchronizing the cell cycle. However, as would be otherwise predictedfrom the art, inducing cells into cell cycling increases the risk of arapidly growing and recurring tumor. But the continued failure of knowncompositions to improve disease-free survival rates or lead to completeremission suggests a need for inducing malignant cells into cell cyclingin a manner that does not proliferate the tumor but increases thesusceptibility of the residual tumor to follow-on treatment withradiation and/or chemotherapy.

Therefore, there is a need for methods for inducing tumor cells into acell cycle selected from the group of (different phases of the cellcycle) G₁, S, G₂ and M where the new methods can be synergisticallyapplied with chemotherapy, immuno-therapy and radiation therapy. Thereis also a need for pre-sensitizing cancer tumors in general along withthe need for a new serum-free and mitogen-free cytokine mixturecomprised of specific ratios of IL-1β to IL-2, TNF-α to IL-2, IFN-γ toIL-2 and GM-CSF to IL-2 that unexpectedly demonstrates far betterefficacy over known compositions in inducing a tumor cells to enter acell cycle phase or for pre-sensitizing a cancer.

SUMMARY OF THE INVENTION

The present invention is based, in part, on methods of pre-sensitizingcancer in general and a new serum-free and mitogen-free cytokine mixturehaving specific ratios of IL-1β to IL-2, TNF-α to IL-2, IFN-γ to IL-2and GM-CSF to IL-2. Accordingly, the present invention enables thedevelopment of compositions useful as a pharmaceutical or as an adjuvantto be used in conjunction with therapeutic cancer treatments such aschemotherapy, immuno-therapy and radiation therapy.

In embodiments of the invention, a method of improving conventionalchemotherapy or radiotherapy of neoplasms or diseases of the immunesystem with a serum-free and mitogen-free cytokine mixture is disclosed.The methods provide for a pre-sensitizing step for the treatment ofcancer in conjunction with radiotherapies or other physical modalitiesof cell killing. A method for inducing tumor cells into a vulnerablecell cycle phase selected from the group of (different phases of thecell cycle) G₁, S, G₂ and M is also contemplated. The present inventionis not limited to any one particular type of cancer and can include anytype of cancer. Specific applications include administering a serum-freeand mitogen-free cytokine mixture peritumorally three times a week overa two week period in a range from about 20 IU to 1600 IU or specificallyat 400 IU or at 800 IU or still further at five times a week in a rangefrom about 20 IU to 1600 IU or at 400 IU or at 800 IU, wherein IUrepresent International Units for Interleukin-2 given in World HealthOrganization 1^(st) International Standard for Human IL-2, 86/504.

Another embodiment includes a serum-free and mitogen-free cytokinepreparation such as Multikine® in novel and non-obvious concentrations.The cytokine preparation may further be part of a pharmaceuticalcomposition. In specific applications, the new serum-free andmitogen-free cytokine preparation has specific ratios of cytokine tointerleukin 2 (IL-2) as follows: IL-1β to IL-2 at a ratio range of0.4-1.5, and preferably at 0.7±0.1 (IL-1β/IL-2), TNF-α to IL-2 at aratio range of 3.2-10.9, and preferably at 9.5±1.8 (TNF-α/IL-2), IFN-γto IL-2 at a ratio range of 1.5-10.9, and preferably at 6.0±1.1(IFN-γ/IL-2), and GM-CSF to IL-2 at a ratio range of 2.2-4.8, andpreferably at 4.0±0.5 (GM-CSF/IL-2).

In other specific applications, the serum-free and mitogen-free cytokinepreparation or pharmaceutical composition has further differentcytokines and other small biologically active molecules in Multikine®wherein the ratio of each of the small biologically active molecules toIl-2 is as follows: IL-3 to Il-2 in a ratio range of 0.38-0.68,preferably at 0.53±0.15, IL-6 to Il-2 in a ratio range of 37.2-53.8,preferably at 46±5.9, IL-8 to Il-2 in a ratio range of 261-561.5,preferably at 41±10.6, IL-1α to Il-2 in a ratio range of 0.56-0.94,preferably at 0.75±0.19, IL-10 to Il-2 in a ratio range of 2.87-3.22,preferably at 3.0±0.18, IL-16 to Il-2 in a ratio range of 1.24-2.94,preferably at 1.84±0.68, G-CSF to Il-2 in a ratio range of 2.16-3.78,preferably at 2.97±0.81, TNF-β to Il-2 in a ratio range of 1.18-2.43,preferably at 1.8±0.63, MIP-1α to Il-2 in a ratio range of 16.78-37.16,preferably at 22.7±7.0, MIP-1β to Il-2 in a ratio range of 19.2-26.4,preferably at 22.8±5.7, a RANTES to Il-2 in a ratio range of 2.3-2.7,preferably at 2.5±0.13, a EGF to Il-2 in a ratio range of 0.27-0.28,preferably at 0.275±0.008, PGE₂ to Il-2 in a ratio range of 3.68-5.42,preferably at 4.5±0.87 and TxB₂ to Il-2 in a ratio range of 23.5-25.1,preferably at 24.3±0.83.

Other objects and advantages of the present invention are set forth inthe following description. The accompanying drawings and tables, whichconstitute a part of the disclosure, illustrate and, together with thedescription, explain the principle of the invention. One of ordinaryskill in the art will appreciate that other aspects of this inventionwill become apparent upon reference to the attached figures and thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be explained in greater detail by the followingdescription and specific embodiments and with the aid of theaccompanying drawings:

FIG. 1 represents the mode of action of Multikine®.

FIG. 2 represents the immuno-histochemistry staining of a tumor in headand neck cancer squamous cell carcinoma with the specific cell cyclemarker Ki-67.

FIG. 3 represents morphometric analysis of Ki-67 immuno-histochemistrystaining of both tumor stroma and tumor epithelia where “*” marks astatistically significant differences where p<0.05 [at α=0.05].

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The present invention is concerned with methods of pre-sensitizingcancer in general and a novel serum-free and mitogen-free cytokinemixture comprised of specific ratios of IL-1β to IL-2, TNF-α to IL-2,IFN-γ to IL-2 and GM-CSF to IL-2. One such novel cytokine mixture isMultikine®, which has demonstrated immuno-modulatory capabilities. Theclinical significance of the immuno-suppression in cancer patientsunexpectedly impacts methods of pre-sensitizing cancer prior totherapeutic treatment and in particular entry of the tumor cell into acell cycle phase.

Immune restoration of head and neck cancer patients is accomplished bythe infusion of cytokines such as IL-2, IFN α-γ or IL-12. Whiteside,“Immunobiology and immunotherapy of head and neck cancer”, Curr OncolRep 2001; 3:46-55. In head and neck cancer, interleukin-based cytokinetherapy resulted in immuno-augmenting. Cortesina G et al.,“Interleukin-2 injected around tumor-draining lymph nodes in head andneck cancer”, Head Neck 1991; 13:125-31; De Stefani et al., “Treatmentof oral cavity and oropharynx squamous cell carcinoma with perilymphaticinterleukin-2: clinical and pathologic correlations”, J Immunother 1996;19:125-33; Valente et al., “Infiltrating leukocyte populations andT-lymphocyte subsets in head and neck squamous cell carcinomas frompatients receiving perilymphatic injections of recombinant interleukin2”, Mod Pathol 1990; 3:702-8; Whiteside T L et al., “Evidence for localand systemic activation of immune cells by peritumoral injections ofinterleukin 2 in patients with advanced squamous cell carcinoma of thehead and neck”, Cancer Res 1990; 53:5654-62; Barrera et al.,“Combination immunotherapy of squamous cell carcinoma of the head andneck”, Arch Otolaryngol Head Neck Surg 2000; 126:345-51; Verastegui etal., “A natural cytokine mixture (IRX-2) and interference with immunesuppression induce immune mobilization and regression of head and neckcancer”, Int J Immunopharmacol 1997; 19:619-27; Hadden et al.,“Interleukins and contrasuppression induce immune regression of head andneck cancer”, Arch Otolaryngol Head Neck Surg 1994; 120:395-403. Forexample, human (r)hIL-2 was successfully used to improve immune functionof head and neck cancer patients as measured by cytotoxic T lymphocyte[CTL] and delayed type hypersensitivity [DTH] responses. The decreasedresponse of T cells was shown by the decreased expression of the T cellreceptor (TCR), its key signaling components, the ζ chain and zap-70,the absence of IL-2 production and increased apoptosis of T cells.Whiteside T L., “Immunobiology and immunotherapy of head and neckcancer”, Curr Oncol Rep 2001; 3:46-55. Studies investigating the causesof the impaired T cell function in head and neck cancer showed that theFas-FasL system, TGF-β and PGE₂ are expressed at high levels.

However, in vivo administration of rIL-2 increased density of CD25⁺cells as well as natural killer (NK) cells, human leukocyte antigen(HLA)-DR⁺ lymphocytes and T cells. In another series of studies,positive clinical responses have been observed when a cytokine mixturewas administered perilymphatically or peritumorally. However, none ofthese studies correlated an increased immune response with definitivefollow-on treatment such as surgery, radiation therapy and/orchemotherapy.

The Technology

Multikine®, a Leukocyte-Interleukin Injection, is a serum-free,mitogen-free, antibiotic-free preparation produced from human peripheralblood mononuclear cells that include T-cells, B cells and macrophages.There are three “families” of cytokines in Multikine® that togetherimpart the unique biological activity of Multikine®. They include directcytotoxic/cytostatic and virocidal/virostatic cytokines such as TNF-α,and IFN-γ, lympho-proliferative cytokines such as IL-1, and IL-2 andchemotactic cytokines such as IL-6, IL-8 and MIP-1α. Furthermore, thedifferent cytokine and small biological molecules that constituteMultikine® are all derived from the lectin (PHA) in vitro stimulation ofhuman peripheral blood mononuclear cells that include T cells, B cells,and macrophages. Centrifugation on a Ficoll-Paque gradient separates thewhite blood cells (including T cells, B cells, and macrophages) fromdonor whole blood, and a series of washes (in physiologically bufferedmedia) facilitates the isolation of lymphocytes, and the removal of redblood cells, cellular debris and other unwanted cellular components fromthe isolated white cell component of the whole donor blood.

Multikine® contains different cytokines present at specific ratios ofeach cytokine to Interleukin 2 (IL-2) as follows: IL-1β to IL-2 at aratio range of 0.4-1.5, and preferably at 0.7±0.1 (IL-1β/IL-2), TNF-α toIL-2 at a ratio range of 3.2-10.9, and preferably at 9.5±1.8(TNF-α/IL-2), IFN-γ to IL-2 at a ratio range of 1.5-10.9, and preferablyat 6.0±1.1 (IFN-γ/IL-2), and GM-CSF to IL-2 at a ratio range of 2.2-4.8,and preferably at 4.0±0.5 (GM-CSF/IL-2).

The remainder of the different cytokines and other small biologicallyactive molecules in Multikine® are also present within each preparationof the small biologically active molecule to Il-2 as follows: IL-3 toIl-2 in a ratio range of 0.38-0.68, preferably at 0.53±0.15, IL-6 toIl-2 in a ratio range of 37.2-53.8, preferably at 46±5.9, IL-8 to Il-2in a ratio range of 261-561.5, preferably at 41±10.6, IL-1α to Il-2 in aratio range of 0.56-0.94, preferably at 0.75±0.19, IL-10 to Il-2 in aratio range of 2.87-3.22, preferably at 3.0±0.18, IL-16 to Il-2 in aratio range of 1.24-2.84, preferably at 1.84±0.68, G-CSF to Il-2 in aratio range of 2.16-3.78, preferably at 2.97±0.81, TNF-β to Il-2 in aratio range of 1.18-2.43, preferably at 1.8±0.63, MIP-1α to Il-2 in aratio range of 16.78-37.16, preferably at 22.7±7.0, MIP-1β to Il-2 in aratio range of 19.2-26.4, preferably at 22.8±5.7, a RANTES to Il-2 in aratio range of 2.3-2.7, preferably at 2.5±0.13, a EGF to Il-2 in a ratiorange of 0.27-0.28, preferably at 0.275±0.008, PGE₂ to Il-2 in a ratiorange of 3.68-5.42, preferably at 4.5±0.87 and TxB₂ to Il-2 in a ratiorange of 23.5-25.1, preferably at 24.3±0.83.

Multikine® was tested using a characterization protocol and does notcontain the following cytokines and other small biologically activemolecules: IL-4, IL-7, and IL-15, TfR, sICAM, PDGF-AB, IFN-α, EPO, LTC4, TGF-β2, FGF basic, Angiogenin, sE-selectin, SCF, and LIF. Multikine®contains only trace quantities (just above the level of detection of theassay) of IL-12, and LTB 4.

In the manufacturing process, mononuclear cells are separated from humandonor “buffy coats” by step-gradient centrifugation and cultured withPHA to enhance production and secretion of IL-2 and other cytokines fromthe donor white blood cells in culture as disclosed in U.S. Pat. Nos.5,093,479, 4,390,623, 4,388,309, 4,406,830, 4,661,447, 4,681,844 and4,464,355, all of which are incorporated herein by reference.Subsequently, the culture supernatant is aseptically harvested,clarified and subjected to a commercial virus exclusion process. Thesupernatant is then further concentrated approximately 10 fold byultrafiltration and microfiltration.

At this point, Human Serum Albumin, Inj. USP is added and theconcentrate is then buffered to a physiological pH and brought to atarget IL-2 concentration per the label claim (example 400 IU/mL). Theconcentrate is then subjected to a second micro-filtration (0.22micron-rated filter) and aseptically dispensed into sterile serum-typevials and labeled by its IL-2 content. Product potency is measured bythe incorporation of radio-labeled thymidine by a cytotoxic T-lymphoidline (CTLL-2). The final injectable agent is further tested by ELISA forthe presence of five marker cytokines: IL-2, IL-1β, GM-CSF, IFN-γ, andTNF-α.

Multikine® is provided frozen in a borosilicate glass serum vialcontaining 2.2 mL of drug at the label claim as IL-2 (400 IU/ml) forperitumoral, intratumoral, perilymphatic or subcutaneous administration.Multikine® is subjected to quality control tests for identity,sterility, bacterial endotoxins, pH, and total protein concentration.Each vial is inspected for particulate contamination and appearance. Thepreparation has a total protein content of 3 mg/mL wherein the materialis supplied sterile and pyrogen free. Multikine® has an assignedexpiration date of 24 months from date of manufacture when the drug isstored at −20° C.

Definitions

IL-2—Interleukin 2 (IL-2): A 15.5-kD glycoprotein synthesized by CD4⁺helper T lymphocytes (Formally known as T cell Growth Factor). IL-2 hasan autocrine effect acting on the CD4+ T lymphocytes that produce it andon other cells of the immune system (including B lymphocytes, CD8+ Tlymphocytes, NK [Natural Killer] cells and others).

IL-1β Interleukin 1 beta (IL-1β): A 17-kD cytokine synthesized byactivated mononuclear phagocytes, is found in free form in thecirculation and mediates inflammatory responses. It acts on CD4+ Tlymphocytes to help facilitate their proliferation, and acts onB-lymphocytes as a growth and differentiation factor. It also inducesthe synthesis of IL-6 by mononuclear phagocytes.

TNF-α—Tumor Necrosis Factor alpha (TNF-α): A 157 amino acid (aa)residues protein, synthesized by stimulated monocytes, macrophages, Blymphocytes, T lymphocytes, an NK cells among others, found in atrimmeric form in the circulation. TNF mediates direct anti-tumoraction, causing tumor cell lysis, facilitates leukocyte recruitment,inducing angiogenesis and promotes fibroblast proliferation.

IFN-γ—Interferon Gamma (IFN-γ): A 21-24-kD glycoprotein homodimersynthesized by activated T lymphocytes and NK cells, is a powerfulactivator of monocytes increasing monocytes ability to destroyintracellular microorganisms and tumor cells. It has direct anti-viraland anti-proliferative activity, and causes many cell types to expressClass II MHC (Major Histocompatibility Complex) cell surface molecularcomplex, as well as increasing the expression of Class I MHC.

GM-CSF—Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF): A 127aa protein found as a monomer in the circulation, produced bymacrophages and T lymphocytes, fibroblast and endothelial cells. It is agrowth factor for hemopoietic cells, and stimulates the growth anddifferentiation of myelomonocytic lineage.

IL-3—Interleukin-3 (IL-3): A 20-kD Lymphokine synthesized by activatedCD4+ T helper lymphocytes, acts as a colony-stimulating factor byfacilitating the proliferation of some hematopoietic cells and promotingthe proliferation and differentiation of T lymphocytes.

IL-6—Interleukin-6 (IL-6): A 26-kD cytokine produced by activated Tlymphocytes, mononuclear phagocytes, endothelial cells, and fibroblasts.It acts on many cells but has a special function in enabling activatedB-lymphocytes to differentiate into antibody secreting plasma cells, andinduces hepatocytes to form acute-phase proteins (implicated ininflammatory responses) as well as fibrinogen.

IL-8—Interleukin-8 (IL-8): An 8-kD protein produced by macrophages andendothelial cells. Is a powerful chemotactic factor for neutrophils andT lymphocytes, and facilitates neutrophil adherence to endothelialcells.

IL-1α—Interleukin 1 alpha (IL-1α): A 17-kD cytokine (like IL-1 iscleaved from a 33-kD precursor molecule, synthesized by activatedmononuclear phagocytes, is rarely found in free form in the circulationand acts as a membrane-associated substance.

It assists IL-1β in mediating inflammatory responses.

IL-10—Interleukin-10 (IL-10): An 18-kD polypeptide produced by CD4+ andCD 8+ T lymphocytes, monocytes, macrophages, activated B lymphocytes,and keratinocytes. It inhibits macrophages ability to present antigenparticularly to T_(H)1-type cells, and secrete IL-6 and TNF.

IL-16—Interleukin-16 (IL-16): A 14-kD tetrameric protein produced byCD8+ T lymphocytes, eosinophils, mast cells and respiratory epithelialcells. It has strong chemoattraction properties for CD4+ T lymphocytesand monocytes.

G-CSF—Granulocyte Colony Stimulating Factor (G-CSF): A 22-25-kDhomodimer glycoprotein produced by macrophages, endothelial cells,fibroblasts and stromal cells. It increases granulocyte progenitor cellsin the marrow, and sustains increase in blood neutrophils. It alsoenhances the ability of neutrophils to exhibit enhanced super-oxideproduction thought to be important in the destruction of microbiallyinfected cells and tumor cells.

TNF-β—Tumor Necrosis Factor beta (TNF-—): A 25-kD protein produced byactivated lymphocytes. It can kill tumor cells in culture, andstimulates proliferation of fibroblasts. In addition it mimics most ofthe other actions of TNF-α.

MIP-1α—Macrophage Inflamatory Protein-1 alpha (MIP-1α): A 66-aamonomeric protein produced by macrophages and other cells. It is achemo-attractant for monocytes, T lymphocytes and eosinophils.

RANTES—An 8-kD protein produced by T lymphocytes and is achemo-attractant to monocytes, T lymphocytes and eosinophils, andpromotes inflammation.

EGF—Epidermal Growth Factor (EGF): A trisulfated polypeptide of 53-aaresidues. EGF is a member of the tyrosin kinase family, and has multiplefunctions including stimulation of the mitogenic response and assistingin wound healing.

PGE₂—Prostaglandin E₂ (PGE₂): PGE₂ belong to a family of biologicallyactive lipids derived from arachidonic acid through the cyclooxygenaseenzymatic reaction. It is released by activated monocytes and blocks MHCClass II expression on T lymphocytes and macrophages.

TxB₂—Thromboxane B₂ (TxB₂): TxB₂ is a member of biologically activecompounds derived from polyunsaturated fatty acids by isomerization ofprostaglandin and endoperoxidase PGH₂ via the enzyme thromboxanesynthetase. TxB₂ has a physiological role in thromboembolic disease, andanaphylactic reactions.

CD25⁺ Cells—CD25 is a single chain glycoprotein, often referred to asthe α-chain of the Interleukin-2-receptor (IL-2R) or the Tac-antigen,that has a mol wt of 55 kDa and is present on activated T and B cellsand activated macrophages. It functions as a receptor for IL2. Togetherwith the β-chain of the IL-2R, the CD25 antigen forms a high-affinityreceptor complex for IL-2.

CTLL-2 (Cell Line)—A line of mouse cytotoxic T lymphocytes obtained fromC57Bl/6 mice. This T cell line is dependent on an exogenous source ofIL-2 for growth and proliferation.

Fas-FasL—The Fas/Fas Ligand system. The combination of a Fas antigen, acell surface transmembrane protein that mediates apoptosis, and acomplementary Fas-activated cytokine on a neutrophil that transduces anapoptotic signal into cells. Fas is a type-I membrane protein belongingto the tumor necrosis factor (TNF) receptor superfamily, and FasL is amember of the TNF family. FAS ligand is a membrane-bound protein of 31kDa [kilo Dalton] (278 amino acids). The Fas-Fas ligand system playsimportant roles in many biological processes, including the eliminationof autoreactive lymphoid cells. The Fas ligand is predominantlyexpressed in activated T lymphocytes and is one of the major effectormolecules of cytotoxic T lymphocytes and natural killer cells.

HLA-DR⁺Lymphocytes—Lymphocytes containing human leukocyte antigen(HLA)-DR antigens, a group of polymorphic glycoproteins determined by aglue sequence found in a leukocyte loci located on chromosome 6, themajor histocompatibility loci in humans.

IU (International Units)—A unit of measure of the potency of biologicalpreparations by comparison to an international reference standard of aspecific weight and strength e.g., WHO 1^(st) International Standard forHuman IL-2, 86/504. International Units are the only recognized andstandardized method to report biological activity units that arepublished and are derived from an international collaborative researcheffort.

U (Units as a measure of biological activity)—Shorthand for a variety ofnamed “units”, which each laboratory derives as a reference, which isfurther unique to the laboratory where the work is being performed. Each“unit” is different from one laboratory to another laboratory and is nota globally recognized standard such as International Units (IU).

Mononuclear Infiltrate—Presence of monocytes, plasma cells, andlymphocytes, in tissue where they “normally” would not be present; orthe presence of these cells in large numbers or abundance in clusterswhere they would otherwise be present in only a small number.

TCR ζ Chain—T-cell receptor-zeta chain. The zeta subunit is part of theTCR complex and is targeted towards the interaction of the TCR cellsurface receptor with its ligand (antigen). The zeta subunit extendinginto the cell cytoplasm (cytosol) is phosphorylated at its tyrosineresidues upon T cell activation and is implicated in signal transductionafter TCR ligation.

TIL (Tumor Infiltrating Lymphocytes)—T lymphocytes isolated from thetumor they are infiltrating. Tumor Infiltrating lymphocytes have littleor no cytotoxicity. TILs include CD4+ CD8+ predominantly T cells, andcan be expanded in vitro by culture in the presence of IL-2. These cellsare activated by the treatment with IL-2 and are frequently moreaggressive towards the tumor from which they were isolated than normallymphokine activated cells. The cytotoxic activity of TILs can beenhanced by IFN-γ. The antitumor activity of TILs in vivo can be blockedby TGF-β.

ZAP 70—A 70 kD Zeta Associated Protein associated with the TCR ζ Chainthat is a tyrosine kinase present in cytosol. ZAP 70 is thought toparticipate in maintaining T lymphocyte receptor signaling, mediatingthe signal transduction which eventually produces IL-2. The ZAP70 geneis expressed in T-cells and natural killer cells and maps to humanchromosome 2q12.

ζ (Zeta) Chain—See TCR ζ Chain—The zeta chain gene is located onchromosome 1 in humans. The extracellular domain of this protein is nineamino acids long whereas the transmembrane domain contains a negativelycharged aspartic acid residue and the cytoplasmic domain is 113 aminoacids long. The cytoplasmic tail contains three of the antigenrecognition motifs found in the cytoplasmic tails of CD3 chains. Thezeta chain is also associated with other receptors such as the Fc(fragment, crystalline)-gamma receptor of NK cells.

USP—U.S. Pharmacopeia Monographs.

P—“p<0.01”: A term in mathematical statistics that denotes the level ofprobability of an event occurring under pre-set conditions.

ANOVA (Analysis of Variances)—A single factor analysis as described inStatistics and mathematical textbooks e.g., “Handbook of StatisticalMethods for Engineers and Scientists”, Harrison M. Wadsworth, Jr., Ed.,McGraw Hill 1990, and “Statistical Operations Analysis of HealthResearch Data”, Robert P. Hirsch and Richard K. Riegelman, Eds.Blackwell Science Inc., 1996.

Mode of action and characterization of Multikine®

Multikine® is a biologically active, minimally toxic, immunomodulatorymixture of naturally derived and naturally occurring human cytokinesproduced under set conditions as described herein. Multikine® can beused as a anti-cancer and anti-viral therapy or as a neo-adjuvanttherapy with a broad-spectrum application for cancer, infectiousdisease, and other diseases states responding to immunomodulation.

Multikine® was developed based on animal studies, which demonstratedthat “mixed interleukins” have immunomodulatory and immunostimulatoryactivity in vitro as shown by Hadden et al., “Mixed Interleukins andThymosin Fraction V Synergistically Induce T Lymphocyte Development inHydrocortisone-Treated Aged Mice”, Cell. Immunol. 144:228-236 (1992).Without being limited to any one theory, it is hypothesized that thelocal/regional injection of “mixed interleukins” such as Multikine®overcomes local immuno-suppression. Subsequently, a break tolerance totumor antigens occurs and allows for an effective local anti-tumorimmune response to occur.

It has been shown that the local instillation of interleukins in theregion of the tumor or the actual transfection of Interleukin genes intoa tumor markedly augments the anti-tumor immune response resulting intumor regression as reported by Golumbek et al., “Treatment ofEstablished Renal Cancer by Tumor Cells Engineered to SecreteInterleukin-4”, Science 254:713-716 (1991). However, none of thesestudies discovered the highly unexpected effect of inducing malignantcells into a cell cycle phase without causing the active proliferationof the tumor.

Quite unexpectedly, the administration of Multikine® pre-surgery leadsto an increase in the number of tumor cells in the cell cycle phasewithout increasing the risk of a more rapidly growing and more rapidlyrecurring tumor as would otherwise be predicted from the art. Theability to induce tumor cells into cell-cycle appears to be unique toMultikine® and may be due to the synergistic effect of the differentcytokines present in this investigational drug and the differentialeffect of these cytokines on both the host's immune system and the tumorcells.

Data regarding the recurrence rate of patients treated with Multikine®prior to surgery showed no recurrence of cancer at 24 months posttreatment with Multikine®. Remarkably, a small cohort of 8 sequentiallytreated patients did not have a single recurring patient in the 24months follow-up period. In stark contrast, the literature teaches therecurrence rate of similar patients at about 50% at 18-24 months postsurgery.

In particular, Multikine® treatment did not appear to induce activeproliferation of tumor residing lymphoid cells. Correspondingly, stromalKi-67⁺ cells decreased while the frequency of Ki-67⁺ cancer cellsincreased following Multikine® treatment. Thus, Multikine® treatmentinduced an increase in the number of cycling tumor cells leading toincreased susceptibility of the residual tumor to follow-on treatmentwith radiation and/or chemotherapy. Although other studies conductedwith both natural and recombinant cytokines have shown efficacy in thetreatment of cancer therapy, those studies have failed to teach theinduction of entry into the cell cycling phase or the new method ofsynergistically combining Multikine® with chemotherapy, immuno-therapyand radiation therapy.

For example, studies with the use of natural human and recombinant IL-2and other cytokines as well as in local-regional therapy demonstratedthe immune augmenting and anti-cancer activity at various sites. Inparticular, IL-2 demonstrates activity in the pleural cavity, liver andthe urinary bladder while IFN-α demonstrates activity in the ovary whileIFN-β demonstrates activity in the brain as reported by Yasumoto et al.,“Induction of lymphokine-activated killer cells by intrapleuralinstillations of recombinant interleukin-2 in patients with, malignantpleurisy due to lung cancer”, Cancer Res 1987; 47:2184-7; Mavilgit etal., “Splenic versus hepatic artery infusion of interleukin-2 inpatients with liver metastases”, J Clin Oncol 1990; 8:319-24; Pizza etal., “Tumor regression after intralesional injection of interleukin-2(IL-2) in bladder cancer. Preliminary report”, Int J Cancer 1984;34:359-67; Berek et al., “Intraperitoneal recombinant α-interferon for“salvage” immunotherapy in stage III epithelial ovarian cancer: agynecologic oncology group study”, Cancer Res 1985; 45:4447-53; andFettel et al., “Intratumor administration of beta-interferon inrecurrent malignant gliomas-A phase I clinical and laboratory study”,Cancer 1990; 65:78-83. Even further, IFN-γ has been shown to demonstrateactivity in skin while TNF-α demonstrates activity in the genitaliawhile a mixture of various cytokines demonstrates activity in the headand neck as reported by Edwards et al., “The effect of intralesionalinterferon gamma on basal cell carcinomas”, J Am Acad Dermatol 1990;22:496-500; Irie et al., “A case of vulva cancer responding to therecombinant human tumor necrosis factor (PT-950) local injectiontherapy”, Gan No Rinsho 1988; 34:946-50; and Pulley et al.,“Intravenous, intralesional and endolymphatic administration oflymphokines in human cancer”, Lymph Res 1986; 5:S157-63. Moreover,studies of recombinant IL-2 administrations for 10 days prior to surgeryin the jugular peri-lymphatic or jugular peri-lymphatic and under thechin showed variable necrosis and lymphocytic infiltration as reportedby Valente et al., “Infiltration leukocyte polulations and T-lymphocytesubsets in head and neck squamous cell carcinomas from patientsreceiving perilymphatic injections of recombinant interleukin-2”, ModPathol 1990; 3:702-8 and DeStefani et al., “Treatment of oral cavity andoropharynx squamous cell carcinoma with perilymphatic interleukin-2:clinical and pathologic correlations”, J Immunother 1996; 19:125-33.Moreover, microscopic examination of the resected tumors demonstrated anincrease in lymphocytic infiltrate correlating to the clinicalobservations of IL-2 as reported by Saito et al., “Immunohistology oftumor tissue in local administration of recombinant interleukin-2 inhead and neck cancer”, Nip Jibi Gakkai Kaiho 1989; 92:1271-6.

Nevertheless, none of the aforementioned studies showed any change inthe gross dimensions of resected tumors despite two remissions atputatively high doses of recombinant IL-2 (800,000 U for four weeks[U=Units]) in 20 head and neck cancer patients as reported by Saito etal., “Clinical evaluation of local administration of rIL-2 in head andneck cancer”, Nip Jibi Gakkai Kaiho. 1989; 921271-6. Furthermore, theaforementioned studies have been limited by the small number of patientsand hampered by the lack of a pathological comparison to a controlgroup.

Although a recent randomized multi-center phase III study of 202 OSCCpatients by De Stefani et al. indicated that peri-lymphaticadministration of low does (5000 U/day [U=Units]) of recombinant humanIL-2 for 10 days prior to surgery, into the ipsylateral cervical lymphnode chain, resulted in a significant (p<0.01) increase in disease-freesurvival, which in turn resulted in longer overall survival (p<0.03), DeStefani et al. failed to assess the role of this treatment regimen oncell cycling and its effect on the improvement of radiation andchemotherapy. See De Stefani et al., “Improved Survival WithPerilymphatic Interleukin 2 in Patients With Resectable Squamous CellCarcinoma of the Oral Cavity and Oropharynx”, Cancer 2002; 95: 90-97.Furthermore, despite teaching 5000 U/day, no comparisons between thepresent invention and De Stefani et al. could be made with regard to DeStefani et al.'s teaching of a “high” and “low” dose of an administeredbiologic because the drug potency was measured by an undefinable U(Units). In contrast, the present invention validated and completed thefull USP analytical methods validation program for determining thebiological activity of Multikine® in IU (International Units).

Methods

Tumor cell proliferation as measured by the immunohistochemistry Ki-67marker, or other equivalent means such as through the use of PCNAmarker, p53 marker were used as a prognostic parameter. de Vicente etal., “Expression of cyclin D1 and Ki-67 in squamous cell carcinoma ofthe oral cavity: clinicopathological and prognostic significance”, OralOncol 2002; 38:301-8; Bettendorf et al., “Prognostic relevance of Ki-67antigen expression in 329 casess of oral squamous cell carcinoma”, ORL JOtorhinolaryngeol Relat Spec 2002; 64:200-5. In conjunction, flowcytometry or conventional staining methods and the use of microscopywith clinical, histopathological and tumor staging and classification(TNM, Tumor, Node, Metastasis) were used with other to indicate theaggressiveness of the disease process. Kerdpon et al., “Expression ofp53 in oral mucosal hyperplasia, dysplasia and squamous cell carcinoma”,Oral Disease 1997; 3:86-92.

In particular, a Ki67 cell proliferation marker differentiates and isspecific for only the cells that are in the cell cycle stages. G₁ is thefirst growth phase; S is the second phase marked by the initiation ofDNA synthesis by the cell where cellular DNA replicates, and G₂ thesecond growth phase of the cell follows DNA replication in which thecell doubles in size. M is the last phase in the cell cycle wheremitosis occurs wherein the cell divides into a daughter cell from theoriginal parent cell. Each resulting cell contains a complete replica ofthe DNA of the original parent cell. Ki67 cellular marker being specificto cells in the cell cycle cannot be found in cells that are in G_(o),which is a resting phase of the cell. During G_(o), the cell does notundergo cellular replication, proliferation or DNA replication. Notably,the cell cycle phase phenomena is a property common to all livingeukaryotic cells including tumor cells.

To detect tumor cell proliferation, the presence of Ki-67 in residualtumor cell nests following surgical excision are determined. Raybaud etal., “Nuclear DNA content, an adjunct to p53 and Ki-67 as a marker ofresistance to radiation therapy in oral cavity and pharyngeal squamouscell carcinoma”, Int J Oral Maxillofac Surg 2000; 29:36-41; Koelbl etal., “p53 and Ki-67 as predictive markers for radiosensitiveity insquamous cell carcinoma of the oral cavity? An immunohistochemical andclinicopathologic study”, Int J Radiat Oncol Biol Phys 2001; 49:147-54.Generally, Ki-67 can be found in cells undergoing cell cycle G₁, S, G₂,and M but not in “resting” tumor cells (G_(o)). Since cycling tumorcells are both more radio- and chemo-sensitive, and non-cycling tumorcells are by-and-large radio- and chemo-resistant.

Accordingly, a study was designed that would analyze byimmuno-histopathology tumors from head and neck cancer patients treatedwith Multikine® prior to surgical resection of the residual tumorfollowed by radiation therapy. Timár et al., “The effect of LeukocyteInterleukin, Injection on the peri- and intratumoral subpopulation ofmononuclear cells and on tumor epithelia—A possible new approach toaugmenting sensitivity to radiation and chemotherapy in oral cancer. Amulti-center Phase I/II clinical trial”, The Laryngoscope [accepted forpublication June 2003]. The study was conducted in a blinded manner andexecuted by three independent qualified pathologists that were blindedto the treatment and patient population, treated or control. Ourclinical study, the results of which are reported here, and areincorporated herein by reference analyzed a cohort of 54 oral squamouscell cancer patients (H&NC) as part of a phase I-II clinical trial.These patients were investigated for safety of the therapeutic regimen,tumor and clinical responses, and for the composition of the mononuclearinfiltrate and cell cycling rates.

Twenty-seven (27) patients of the 54 patients cohort receivedperitumoral administration of Multikine® in a dose escalating study.This study resulted in the demonstration that the pre-treatment of headand neck cancer patients with Multikine® tumor induced entry into cellcycle phase, G₁, S, G₂, and M, but not G_(o). This lead to a decrease inrecurrence rate and an increase in disease-free survival of Multikine®treated patients.

In our study, Multikine® administrations were performed in the followingmanner: daily dose was injected peritumorally over a two-week period (3times per week) at the following doses for each of the dose groupstested; low dose, 400 IU (International Units of IL-2) [IL-2-equivalent]daily (8 patients), medium dose, 800 IU (IL-2-equivalent) daily (12patients), and 5 times per week at the high dose, 800 IU(IL-2-equivalent) daily (7 patients). All Multikine® injections wereadministered intra-dermally at the circumferential margin of thevisible/palpable tumor mass. Surgery aimed at resection of the residualtumor mass was performed between day 21 and day 28 following the initialadministration of Multikine®. Local/regional radiation therapy commencedin post-operative patients following wound healing at variable timespost-surgery and was dependent on the individual patient recovery fromthe surgical intervention. Radiotherapy was generally initiated betweentwo to four weeks post-surgery.

The administration of Multikine® was preceded by the single intravenousinfusion of cyclophosphamide, 300 mg/m² three-days prior to the firstMultikine® administration. Indomethacin (25 mg) was self-administeredorally (with food), three times daily, beginning 3 days postcyclophosphamide administration and until 24 hours prior to surgery.Zinc sulfate (50 mg) and multivitamin supplement, once daily, wasself-administered beginning 3 days after cyclophosphamide administrationand until 24 hours prior to surgery. The patients were counseled andencouraged to continue self-administration of multivitamin and Zincregimen following surgery. These agents have no effect whatsoever ontumor cell cycling and were given at doses that are 3-5 fold bellow thenormal cancer therapeutic doses for these drugs.

Results

Detection of cycling cells by Ki-67 expression identified cancer cellsas shown in FIG. 1 and stromal cells (host cells: mononuclear cells,fibroblasts, endothelial cells etc.). Morphometric analysis of thedensity of Ki-67⁺ cancer cells indicated that Multikine® treatmentinduced significant increase (p<0.05) in cycling tumor cells except atthe highest Multikine® dose administered as shown in FIG. 2. On theother hand, the incidence of cycling host cells found primarily in thestromal area of the tumor decreased with an increasing Multikine® doseagain shown in FIG. 2. Effects were proved to be significant for thelowest and the highest doses (p<0.05). These findings support theconclusion that treatment with Multikine® treatment causes cancer cellsto enter a cell cycle phase but does not cause the host immune cells orstromal cells to cycle.

Accordingly, the present invention contemplates Multikine® treatment toinduce cell cycle entry of a high proportion of the tumor cellpopulation based on the expression of Ki-67 antigen.

As stated herein, preliminary data regarding the recurrence rate ofpatients treated with Multikine® prior to surgery, which were eitherfollowed by radiation therapy or watchful waiting, did not exhibit anincrease in the recurrence rate at 24 months post treatment withMultikine®. A small cohort of 8 sequentially Multikine® treated patientsdid not have a single recurring patient in the 24 months follow-upperiod. In contrast, the literature pegs the recurrence rate of thesepatients at about 50% at 18-24 months post surgery.

Moreover, Multikine® treatment did not appear to induce activeproliferation of tumor residing lymphoid cells, and correspondinglystromal Ki-67⁺ cells decreased, while the frequency of Ki-δ67 ⁺ cancercells increased following Multikine® treatment. Thus, Multikine®treatment induced the increase the number of cycling tumor cells leadingto increased susceptibility of the residual tumor to follow-on treatmentwith radiation and/or chemotherapy.

Treatment Regimen with Multikine® for Cancer

The treatment regimen for the pre-sensitization of cancer withMultikine® is predicated on treatment protocol developed for head andneck cancer patients, which has been proven in a statisticallysignificant manner to significantly increase tumor cell cycling aimed atrendering these tumor cells more sensitive to follow on treatment withradiation and/or chemotherapy.

The treatment will include the administration of Multikine®subcutaneously in the area of the submandibular cervical lymph nodechain.

A two-week course of ten (10) subcutaneous/subdermal daily injections ofMultikine® at a daily dose ranging from about 20 IU to 1600 IU as IL-2and will be administered ½ peritumorally at the circumferential marginof the tumor mass, and ½ at the submandibular lymphatic chainipsylateral to the tumor mass. Another course of treatment canpreferably in the range of 40 IU to 800 IU. Still another range can bein the range of 35 IU to 75 IU.

One specific non-limiting example of a suggested treatment contemplatesadministration of Multikine® at a daily dose of 55 IU as IL-2 in atwo-week course of ten (10) subcutaneous/subdermal daily injections

Drug Safety, Pilot Efficacy and Compositions

Multikine® has been tested in over 190 Cancer, HIV, and HIV/HPVinfected, patients with no severe adverse events related to Multikine®administration as reported by Harris et al., “Immunologic approaches tothe treatment of prostate cancer”, Semin Oncol. August 1999;26(4):439-7; Timár et al., “The effect of Leukocyte Interleukin,Injection on the peri- and intratumoral subpopulation of mononuclearcells and on tumor epithelia—A possible new approach to augmentingsensitivity to radiation and chemotherapy in oral cancer. A multi-centerPhase I/II clinical trial”, The Laryngoscope [accepted for publicationJune 2003]; Brown et al., “A Phase I Open-Label Study of LeukocyteInterleukin, Injection in HIV-1 infected individuals: preliminaryevidence for improved delayed-type hypersensitivity responses to recallantigens”, Antiviral Therapy 5 (supplement) 18, 2000; Taylor et al.,“Immunotherapy with Leukocyte Interleukin, Injection for human papillomavirus (HPV) induced cervical dysplasia in HIV patients”, Annual Meetingof the International Society for Interferon and Cytokine Research,Cleveland, Ohio, October 2001; Taylor et al., “Immunotherapy withLeukocyte Interleukin, Injection for human papilloma virus (HPV) inducedcervical dysplasia in HIV patients”, 33rd SGO Conference, Miami, Fla.,March 2002 Multikine® was also shown to be safe in animal toxicologicalstudies in mice, rats, guinea pigs and dogs. Furthermore, Multikine® wastested for and has demonstrated pilot efficacy in head and neck cancerand cervical dysplasia.

Multikine® may further be used as a component of an immunomodulatorycomposition together with one or more pharmaceutically acceptablecarriers or adjuvants, either prophylactically or therapeutically. Whenprovided for use prophylactically, the immunomodulatory composition isprovided in advance of any evidence of infection or disease. While it ispossible for Multikine® to be administered in a pure or substantiallypure form, a pharmaceutical composition, formulation or preparation mayalso be used.

The formulations of the present invention, both for clinical and forhuman use, comprise Multikine® as described above together with one ormore pharmaceutically acceptable carriers and, optionally, othertherapeutic ingredients, especially therapeutic immunological adjuvants.The carrier(s) must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not deleterious to therecipient thereof.

In general, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, bringingthe product into the desired formulation. The term “pharmaceuticallyacceptable carrier” as used herein refers to any carrier, diluent,excipient, suspending agent, lubricating agent, adjuvant, vehicle,delivery system, emulsifier, disintegrant, absorbant, preservative,surfactant, colorant, flavorant, or sweetener. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethod well-known in the pharmaceutical art.

Formulations suitable for intravenous, intramuscular, subcutaneous, orintraperitoneal, nasal, etc. administration conveniently comprisesterile aqueous solutions of the active ingredient(s) with solutionswhich are preferably isotonic with the blood of the recipient. Thecompounds of the present invention may also be administered orally,parenterally, by inhalation spray, topically, rectally, buccally,vaginally or via an implanted reservoir in dosage formulationscontaining conventional non-toxic pharmaceutically-acceptable carriers,adjuvants and vehicles. The term parenteral as used herein includessubcutaneous, intravenous, intramuscular, intraperitoneally,intrathecally, intraventricularly, intrasternal and intracranialinjection or infusion techniques.

Such formulations may be conveniently prepared by dissolving solidactive ingredients in water containing physiologically compatiblesubstances such as sodium chloride (e.g. 0.1-2.0M), glycine, and thelike, and having a buffered pH compatible with physiological conditionsto produce an aqueous solution and rendering the solution sterile. Thesemay be present in unit or multi-dose containers, for example, sealedampules or vials.

The compounds of the present invention may also be administered in theform of sterile injectable preparations, for example, as sterileinjectable aqueous or oleaginous suspensions. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparations may also be sterile injectable solutions orsuspensions in non-toxic parenterally-acceptable diluents or solvents,for example, as solutions in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as solvents or suspending mediums. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids such as oleic acid and its glyceridederivatives, including olive oil and castor oil, especially in theirpolyoxyethylated versions are useful in the preparation of injectables.These oil solutions or suspensions may also contain long-chain alcoholdiluents or dispersants.

The compounds of this invention may also be administered topically,especially when the conditions addressed for treatment involve areas ororgans readily accessible by topical application including disorders ofthe eye, the skin, or the lower intestinal tract. Suitable topicalformulations are readily prepared for each of these areas.

For topical application to the eye, or ophthalmic use, the compounds canbe formulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, the ophthalmic uses of Multikine® may beformulated in an ointment such as petrolatum.

For topical application to the skin, the compounds can be formulated ina suitable ointment containing the compound suspended or dissolved in,for example, a mixture with one or more of the following: mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylenepolyoxypropylene compound, emulsifying wax and water. Alternatively, thecompounds can be formulated in a suitable lotion or cream containing theactive compound suspended or dissolved in, for example, a mixture of oneor more of the following: mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Some factorsinclude the activity of the specific compound employed, the age, bodyweight, general health, sex, and diet of the patients; the time ofadministration, rate of excretion, drug combination, and the severity ofthe particular disease being treated and form of administration.

Pharmaceutical methods may also be employed to control the duration ofaction. Controlled release preparations may be achieved through the useof polymer to complex or absorb the peptide. The controlled delivery maybe exercised by selecting appropriate macromolecules (for example,polyester, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe methods of incorporation in order to control release.

For example, Multikine® may be incorporated into a hydrophobic polymermatrix for controlled-release over a period of days. Suchcontrolled-release films are well known to the art. Particularlypreferred are transdermal delivery systems. Other examples of polymerscommonly employed for this purpose that may be used in the presentinvention include non-degradable ethylene-vinyl acetate copolymer anddegradable lactic acid-glycolic acid copolymers which may be usedexternally or internally. Certain hydrogels such aspoly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful,but for shorter release cycles then the other polymer releases systems,such as those mentioned above.

Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacrylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions.

To be effective therapeutically as central nervous system targets,Multikine® should also readily penetrate the blood-brain barrier whenperipherally administered. Compounds which cannot penetrate theblood-brain barrier can be effectively administered by anintraventricular route or other appropriate delivery system suitable foradministration to the brain.

Multikine® may also be supplied in the form of a kit, alone, or in theform of a pharmaceutical composition as described above. Administrationof Multikine® can be conducted by conventional methods. For example,Multikine can be used in a suitable diluent such as saline or water, orcomplete or incomplete adjuvants. Multikine® can be administered by anyroute appropriate for immune system stimulation, such as intravenous,intraperitoneal, intramuscular, subcutaneous, nasal, oral, rectal,vaginal, and the like.

As noted above, Multikine® may be for either a prophylactic ortherapeutic purpose. When provided prophylactically, Multikine® isprovided in advance of any evidence or in advance of any symptom due todisease. When provided therapeutically, Multikine® is provided at (orafter) the onset of the disease or at the onset of any symptom of thedisease. The therapeutic administration of Multikine® serves toattenuate the disease and improves conventional treatment outcomes.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

1. A method for pre-sensitizing cancer prior to a therapeutic treatment, comprising the step of: administering a therapeutically active amount of a serum-free and mitogen-free cytokine mixture to cancer.
 2. The method of claim 1, wherein said therapeutic treatment is selected from the group consisting of chemotherapy, immuno-therapy and radiation therapy.
 3. The method of claim 1, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period in a range from about 20 IU to 1600 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 4. The method of claim 1, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period in a range from about 40 to 800 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 5. The method of claim 1, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period in a range from about 35 IU to 75 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 6. The method of claim 1, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period at 55 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 7. The method of claim 1, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period at 400 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 8. The method of claim 1, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period at 800 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 9. The method of claim 1, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered five times a week over a two week period at 800 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 10. The method of claim 1, wherein said serum-free and mitogen-free cytokine mixture is comprised of specific ratios of cytokines selected from the group of IL-1β, TNF-α, IFN-γ and GM-CSF to Interleukin-2 (IL-2) as follows: IL-1β to IL-2 at a ratio range of 0.4-1.5; TNF-α to IL-2 at a ratio range of 3.2-10.9; IFN-γ to IL-2 at a ratio range of 1.5-10.9; and GM-CSF to IL-2 at a ratio range of 2.2-4.8.
 11. The method of claim 10, wherein said specific ratios of cytokines are as follows: IL-1β to IL-2 at a ratio range of 0.6 to 0.8; TNF-α to IL-2 at a ratio range of 7.7 to 11.3; IFN-γ to IL-2 at a ratio range of 4.9 to 7.1; and GM-CSF to IL-2 at a ratio range of 3.5 to 4.5.
 12. The method of claim 1 wherein the serum-free and mitogen-free cytokine mixture is Multikine®.
 13. A method for inducing tumor cells into a cell cycle selected from the group of G₁, S, G₂ and M, comprising the step of: administering a therapeutically active amount of a serum-free and mitogen-free cytokine mixture to a cancerous cell.
 14. The method of claim 13, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period in a range from about 20 IU to 1600 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 15. The method of claim 13, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period in a range from about 40 IU to 800 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 16. The method of claim 13, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period in a range from about 35 IU to 75 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 17. The method of claim 13, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period at 55 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 18. The method of claim 13, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period at 400 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 19. The method of claim 13, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered three times a week over a two week period at 800 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 20. The method of claim 13, wherein said serum-free and mitogen-free cytokine mixture is peritumorally administered five times a week over a two week period at 800 IU wherein IU represent International Units for Interleukin-2 given in World Health Organization 1^(st) International Standard for Human IL-2, 86/504.
 21. The method of claim 13, wherein said serum-free and mitogen-free cytokine mixture is comprised of specific ratios of cytokines selected from the group of IL-1β, TNF-α, IFN-γ and GM-CSF to Interleukin-2 (IL-2) as follows: IL-1β to IL-2 at a ratio range of 0.4-1.5; TNF-α to IL-2 at a ratio range of 3.2-10.9; IFN-γ to IL-2 at a ratio range of 1.5-10.9; and GM-CSF to IL-2 at a ratio range of 2.2-4.8.
 22. The method of claim 21, wherein said specific ratios of cytokines are as follows: IL-1β to IL-2 at a ratio range of 0.6 to 0.8; TNF-α to IL-2 at a ratio range of 7.7 to 11.3; IFN-γ to IL-2 at a ratio range of 4.9 to 7.1; and GM-CSF to IL-2 at a ratio range of 3.5 to 4.5.
 23. The method of claim 13 wherein the serum-free and mitogen-free cytokine mixture is Multikine®.
 24. A serum-free and mitogen-free cytokine mixture, comprising specific ratios of cytokines selected from the group of IL-1β, TNF-α, IFN-γ and GM-CSF to Interleukin-2 (IL-2) as follows: IL-1β to IL-2 at a ratio range of 0.4-1.5; TNF-α to IL-2 at a ratio range of 3.2-10.9; IFN-γ to IL-2 at a ratio range of 1.5-10.9; and GM-CSF to IL-2 at a ratio range of 2.2-4.8.
 25. The serum-free and mitogen-free cytokine mixture of claim 24, wherein said specific ratios of cytokines are as follows: IL-1β to IL-2 at a ratio range of 0.6 to 0.8; TNF-α to IL-2 at a ratio range of 7.7 to 11.3; IFN-γ to IL-2 at a ratio range of 4.9 to 7.1; and GM-CSF to IL-2 at a ratio range of 3.5 to 4.5.
 26. A pharmaceutical composition for use in treating cancer, comprising specific ratios of cytokines selected from the group of IL-1, TNF-α, IFN-γ and GM-CSF to Interleukin-2 (IL-2) as follows: IL-1β to IL-2 at a ratio range of 0.4-1.5; TNF-α to IL-2 at a ratio range of 3.2-10.9; IFN-γ to IL-2 at a ratio range of 1.5-10.9; GM-CSF to IL-2 at a ratio range of 2.2-4.8, and optionally in combination with a pharmaceutically acceptable excipient, carrier or additive.
 27. The pharmaceutical composition of claim 26, wherein said specific ratios of cytokines are as follows: IL-1β to IL-2 at a ratio range of 0.6 to 0.8; TNF-α to IL-2 at a ratio range of 7.7 to 11.3; IFN-γ to IL-2 at a ratio range of 4.9 to 7.1; and GM-CSF to IL-2 at a ratio range of 3.5 to 4.5.
 28. The pharmaceutical composition of claim 27, further comprising an IL-3 to IL-2 ratio in a range from 0.38-0.68, preferably at 0.53±0.15
 29. The pharmaceutical composition of claim 27, further comprising an IL-6 to IL-2 ratio in a range from 37.2-53.8, preferably at 46±5.9.
 30. The pharmaceutical composition of claim 27, further comprising an IL-8 to IL-2 ratio in a range from 261-561.5, preferably at 41±10.6.
 31. The pharmaceutical composition of claim 27, further comprising an IL-1α to IL-2 ratio in a range from 0.56-0.94, preferably at 0.75±0.19.
 32. The pharmaceutical composition of claim 27, further comprising an IL-10 to IL-2 ratio in a range from 2.87-3.22, preferably at 3.0±0.18.
 33. The pharmaceutical composition of claim 27, further comprising an IL-16 to IL-2 ratio in a range from 1.24-2.84, preferably at 1.84±0.68.
 34. The pharmaceutical composition of claim 27, further comprising a G-CSF to IL-2 ratio in a range from 2.16-3.78, preferably at 2.97±0.81.
 35. The pharmaceutical composition of claim 27, further comprising a TNF-β to IL-2 ratio in a range from 1.18-2.43, preferably at 1.8±0.63.
 36. The pharmaceutical composition of claim 27, further comprising a MIP-1α to IL-2 ratio in a range from 16.78-37.16, preferably at 22.7±7.0.
 37. The pharmaceutical composition of claim 27, further comprising a MIP-1β to IL-2 ratio in a range from 19.2-26.4, preferably at 22.8±5.7.
 38. The pharmaceutical composition of claim 27, further comprising a RANTES to IL-2 ratio in a range from 2.3-2.7, preferably at 2.5±0.13.
 39. The pharmaceutical composition of claim 27, further comprising a EGF to IL-2 ratio in a range from 0.27-0.28, preferably at 0.275±0.008.
 40. The pharmaceutical composition of claim 27, further comprising a PGE₂ to IL-2 ratio in a range from 3.68-5.42, preferably at 4.5±0.87.
 41. The pharmaceutical composition of claim 27, further comprising a TxB₂ to IL-2 ratio in a range from 23.5-25.1, preferably at 24.3±0.83. 